The inflammatory response governs a wide range of illness from injury to infections and allergies. Initiating inflammation involves activating immune cells that trigger the phospholipase A2 (PLA2)-involved inflammatory processes. PLA2 enzymes are a diverse family of enzymes that hydrolyze the sn-2 fatty acyl bond of phospholipids to produce, among other things, arachidonic acid (AA). They have a wide range of functions involving dietary phospholipid digestion, cellular phospholipid metabolism and turnover, membrane phospholipid remodeling, and critical roles in the inflammatory processes. PLA2 enzymes are abundant in pancreatic juice and venoms of snakes and bees. They are also present in small amounts in many types of cells, including immune cells.
Three types of PLA2 have been found in mammalian tissues: secretory PLA2 (sPLA2); cytosolic PLA2 (cPLA2); and the calcium-independent PLA2 (ciPLA2). sPLA2 hydrolyzes the fatty acyl group at the sn-2 position of phospholipids at the air/water interface. They require millimolar calcium for their enzymatic reactions. sPLA2 has been found to correlate with local and systemic inflammatory responses (1). For example, high levels of sPLA2 have been found in the plasma of patients with acute sepsis, in synovial fluids from patients with arthritis, and in peritoneal fluids from patients with peritonitis.
sPLA2 
sPLA2 enzymes have been implicated in human diseases, particularly in inflammatory diseases including COPD, cystic fibrosis and sepsis. However, the precise function of sPLA2 is not clear. For instance, it is not clear how sPLA2 enzymes exert their action on cells without indiscriminately destroying the cells.
At least ten sPLA2 isoforms have been identified in humans, each with molecular weights around 14 kDa (2-4). The various isoforms of sPLA2 have the same catalytic reactions in terms of phospholipid hydrolysis, i.e., hydrolyzing the fatty acyl group at the sn-2 position of phospholipids at the air/water interface. All sPLA2 require millimolar calcium for enzymatic reactions and interact strongly with membranes containing anionic phospholipids but interact weakly with an interface composed of zwitterionic phosphatidylcholine (PC). Isoforms sPLA2-IB and sPLA2-IIA have been most extensively studied. sPLA2-IB is considered a pancreatic enzyme whose function mainly involves digestion of dietary phospholipids. sPLA2-IIA is a non-pancreatic enzyme and has been found to correlate with local and systemic inflammatory responses (5). sPLA2-IIA is present in platelets and inflammatory cells including neutrophils and has been found in circulating blood and rheumatoid arthritic synovial fluid (5-7). The primary structure of human sPLA2-IIA in platelets and synovial fluid has been determined and its gene cloned (7, 8).
Both sPLA2-IB and sPLA2-IIA have been implicated in human diseases, particularly in inflammatory diseases (9). High levels of sPLA2-IIA have been found in the plasma of patients with acute sepsis, in synovial fluids from patients with arthritis, and in peritoneal fluids from patients with peritonitis (7, 9). sPLA2-IIA may also act as an antibacterial agent to destroy bacteria during infection (10) due to the high cationic charge of sPLA2-IIA (pI>10.5) that, in conjunction with bactericidal/permeability-increasing protein, enables sPLA2-IIA to readily penetrate the cell wall of gram-negative bacteria and disrupt the anionic bacterial membrane.
Inhibiting sPLA2 production has long been considered for therapeutic purposes (11). However, conventional drugs developed to inhibit sPLA2 production or to restrain PLA2 activity have serious side effects and sometimes even exacerbate the pathological conditions. This is, in part, because the complexity of PLA2 enzymes makes drug design for detecting, treating and preventing inflammatory disease more difficult (12).
Conventionally, PLA2 activity is measured by methods that involve the use of radioactive materials, which are inconvenient, time-consuming and biohazardous. A fluorescent liposome-based method has been described but the method is of low sensitivity in comparison to the radioactive methods (13). Another available fluorescence method incorporates fluorescent bis-BODIPY FL C11-PC into the cellular membrane; however, it can only measure the PLA2 activity indirectly (14). Other prior art methods include the pH titration method and the monolayer method, both of which require bulk volumes of reaction solutions, substrates and enzymes.
Therefore, a need exists for an efficient method for detecting, inhibiting and preventing sPLA2 activity in a controlled, non-invasive manner to treat or prevent specific diseases.
Albumin
It has long been shown that some serum proteins including albumin can affect the activity of sPLA2 in the in vitro assay. Albumin possesses dual effects on sPLA2 activity, either stimulating or inhibiting sPLA2 activity, depending on the assay conditions (15). It is generally believed that albumin stimulates sPLA2 activity by removing the PLA2-generated product lyso phospholipids, and inhibits sPLA2 activity by binding the substrates, particularly with low concentrations of substrate liposomes, or removing negatively charged fatty acid from the enzyme/substrate interface (15).
Human serum albumin, a heart-shaped protein, consists of 585 amino acid residues with a calculated molecular weight of 66,439 and a pI value of 5.2. Albumin constitutes more than 60% of total blood plasma protein and plays important roles in fluid distribution throughout the body because of its colloidal properties, in acid-base physiology because of its unique composition and abundance, and in transport because of its high ligand-binding affinity. Although albumin is a monomeric protein, it is organized into three homologous domains (labeled I-III) and each domain is comprised of two sub-domains (A and B) which share common structural elements (16, 17). Its diverse bound-ligands and potential subjection to oxidation of its high content of disulphide bridges, albumin is considered to consist of heterogeneous forms that can be fractionated by passing through an anionic exchange column (18).
An array of different drugs have been found to bind to albumin with great affect on the pharmacokinetics of the drugs (19). Most of the associations between albumin and the bound-substances involve albumin's hydrophobic interaction property. In disease and malnutrition, the quantity and quality of albumin in the circulating blood are diminished. Changes in albumin quantity and quality not only affect on albumin's multiple roles, it may also have a consequence on drug transport efficacy and elimination mechanisms (20). Although the quantity of albumin in the plasma is widely determined by the bromocresol dye methods in clinical laboratories (21, 22), the quality change of albumin such as its binding or interaction properties with PLA2 substrates or products in the blood cannot be simply determined.
Chronic Obstructive Pulmonary Disease
Chronic obstructive pulmonary disease (COPD) is a complex group of conditions associated with progressive airway obstruction and loss of lung function. Two major respiratory disorders associated with COPD—chronic bronchitis and emphysema—damage the lungs and make it difficult for air to move in and out of the lungs and for normal gas exchange to occur. Typical symptoms include shortness of breath, chronic cough and dyspnea on exertion. These symptoms worsen during periods of exacerbation that are typically caused by viral or bacterial infections but may be triggered for other reasons. Patients suffering from COPD often exhibit an increased level of sPLA2-mediated inflammation.
Approximately eleven percent of the United States population, both diagnosed and undiagnosed, suffer from COPD. COPD is the fourth leading cause of death in the United States, and the cost of caring for patients with COPD is estimated to be as high as $40 billion annually (23).
Existing diagnostic methods for detecting and characterizing COPD include pulmonary function testing, pulse oximetry, radiological procedures and monitoring arterial blood gases. However, such testing only picks up relatively advanced cases of COPD and may not detect subtle abnormalities in individuals who have early or mild disease.
COPD treatments are not curative and are mainly focused on palliative care and preventing disease progression and complications. Current treatments include smoking cessation, prevention and management of infections, antioxidant supplementation, vaccinations, life style changes (i.e. avoiding exposure to inhaled irritants), pulmonary rehabilitation, medications (bronchodilators and corticosteroids) and lung transplantation.
Slowing disease progression is currently the objective of most treatments. However, successfully halting or slowing COPD progression is predicated upon early diagnosis and intervention. Currently, there is no reliable way to predict which individuals will develop COPD or which patients with COPD will become progressively worse and develop severe respiratory dysfunction. Efforts to develop a method to monitor the level of inflammation and oxidative stress present in patients with COPD, especially during periods of exacerbation, continue. These efforts involve invasive testing to monitor biomarkers such as carbon monoxide (CO) levels and noninvasive measures of CO, nitric oxide, and other oxidants and cytokines using expired breath condensates. A recent study of screening using an array of 36 systemic biomarkers for assessing COPD exacerbation found that those systemic biomarkers were not helpful in predicting exacerbation severity. The most selective biomarker was C-reactive protein (CRP). However, this was neither sufficiently sensitive nor specific by itself.
Therefore, a need exists for a non-invasive method of diagnosing and monitoring the subtle, sPLA2-mediated inflammation associated with COPD.
Cystic Fibrosis
Cystic fibrosis (CF) is a lung disease characterized by bacterial infection and intense inflammation that is often fatal. CF is caused by the defect of the gene encoding the CF transmembrane conductance regulator (CFTR), a large, membrane-spanning protein that regulates ion flux through the apical surfaces of epithelial cells. Pulmonary complications due to progressive bronchiectasis are the major cause of morbidity and mortality of the CF patients (24). Lower respiratory tract secretions of most CF patients contain high amounts of proteases, particularly the elastase from polymorphonuclear neutrophils (PMN). The abundant neutrophil elastase (NE) is thought to be a major cause of the epithelial tissue damage that leads to bronchiectasis and bronchial obstruction (25, 26).
It has long been recognized that elevating levels of AA in the lungs of patients with CF is linked to the pathogenesis of chronic lung inflammation (27). High arachidonic acid (AA) levels are also associated with phospholipids in lung tissue of CFTR gene knockout cftr−/−-mice (28), and high levels of AA have been linked to low amounts of phospholipid-bound docosahexaenoic acid (DHA) in involved tissues (29). Epithelial cell lines with the deltaF508 mutation in their CFTR gene also released abnormally high levels of AA when induced by Ca2+ (29).
Little is known about the regulation of the production of the high level of AA and the synthesis of the lipid mediators in the CF lung and airway. However, it appears that a cycle of enhanced LTB4 production from AA, chemoattraction of neutrophils, and intense inflammation due to neutrophil flux into lung tissue occurs and stimulates and sustains chronic inflammation (and progressive damage) in the CF lung. Also, the function of surfactant in the CF lung is impaired, and the surfactant phospholipid level is low. All these suggest that PLA2-mediated inflammation may play a critical role in the CF lung injury.
To investigate whether the increase in AA in bronchial secretions of CF patients is due to the increase in PLA2 activity, the inventors previously discovered that bronchealveolar lavage fluid (BALF) from subjects with CF markedly induced PLA2 activity in vitro (U.S. Pat. No. 6,180,596) (30). This revealed that there might be a PLA2 stimulating factor in the BALFs of CF subjects.
Therefore, a need exists for a non-invasive method of diagnosing, monitoring and preventing the sPLA2-mediated inflammation associated with CF.
Sepsis
Infections are the most common causes of late deaths in trauma patients and a frequent cause of morbidity and mortality in hospitalized patients. Infected patients are at risk of developing sepsis, a systemic inflammatory response which causes a widespread and overwhelming activation of the immune system. Severe sepsis leads to tissue deterioration and multi-organ failure.
In the United States, sepsis is the 10th most common cause of death with the incidence of sepsis and sepsis-related deaths increasing by 1.5% per year (31). Recently, it was estimated that $16.7 billion in total national hospital cost in the United States is invoked by severe sepsis; this is based on 751,000 severe sepsis cases per year with 215,000 associated deaths annually. In the last decade, available therapies have been unsuccessful in significantly reducing the mortality rate from sepsis.
Early detection and diagnosis of sepsis is one of the most critical factors in determining patient outcome. Unfortunately, in early stages of sepsis, symptoms including sPLA2-mediated inflammation are often subtle and non-specific, and warning signs, if present, can be easily overlooked or misdiagnosed. By the time the symptoms are obvious, treatment becomes much more challenging, and the likelihood of a successful outcome declines.
Thus, accurate early detection of evolving sepsis in the at-risk patient is a key to the successful treatment of sepsis and lowering the considerable mortality rates that are associated with sepsis. Therefore, a need exists for a non-invasive method of diagnosing, monitoring and preventing the sPLA2-mediated inflammation associated with sepsis.